This project is designed to characterize the structure, function, and regulation of the "classical" reduced folate carrier (RFC) which mediates uptake of reduced folate cofactors into human cells. Adequate supplies of folates are essential for normal growth, function, and development of all tissues and there is growing evidence that folate deficiency contributes to chromosomal instability and malignant transformation. RFC is also critical to the antitumor effectiveness of methotrexate (MTX) and related antifolate chemotherapeutic drugs, and defects in the expression and function of RFC are key factors in the development of MTX resistance. However, until recently, the molecular characterization of RFC was limited, in large part due to the lack of critical reagents (antibodies, cDNAs) for studying this system in human cells. The investigation outlined in this application will expand on recent advances made by our laboratory including the isolation and characterization of multiple human RFC cDNAs arising from heterogeneous transcripts, the determination of the organization and structure of the human RFC gene, and the demonstration of dual promoters and identification of putative cis regulatory elements. Sensitive antibodies to human RFC were prepared and verified, and studies were begun to characterize the molecular alterations responsible for transport- mediated MTX resistance, and the functional importance of key amino acids (Asn58, Gly44, Ser127) in RFC by site-directed mutagenesis. For the continuation of this project, we will (Aim 1) verify computer models of RFC membrane topology by epitope insertion mutagenesis, and establish the structural and functional elements of the RFC protein by affinity labeling and site-directed and random mutagenesis. We will (Aim 2) express epitope-tagged RFC in transport-impaired human cells under control of an inducible promoter, in order to identify changes in transport function with carrier expression, and the roles of protein interactions with RFC in regulating transport function. Additional studies will focus on the role of RFC phosphorylation in regulating folate and antifolate transport. We will (Aim 3) further characterize the cis regulatory elements in the dual RFC promoters and investigate how changes in promoter utilization or RNA splicing involving upstream non-coding exons regulate RFC levels and function in normal tissues and tumors. The knowledge gained from these experiments will be used to characterize the mechanisms of p53- mediated downregulation of RFC gene expression, and how alterations at both the transcriptional and posttranscriptional levels result in decreased RFC expression and impaired MTX transport in drug resistant tumors. Collectively, our studies should clarify the structural and regulatory determinants of RFC expression, the protein determinants of folate and antifolate substrate binding and membrane translocation, and the molecular bases of impaired RFC function in MTX resistant tumor cells.